6 steps to compare the performance of two different reaction mixtures for real-time PCR

# Real-Time PCR Mix Evaluation Guide When evaluating a new real-time PCR master mix, it's crucial to consider the right parameters. This guide offers a protocol along with tips on quickly assessing a real-time PCR master mix. --- **Real-Time PCR Mix Evaluation Guide** --- ## Step-by-Step Evaluation Protocol ### 1. Prepare a DNA Dilution Series Create a DNA dilution series following the pattern shown below. Gently mix the tubes by flicking them (do **not vortex!**) and centrifuge briefly.  *Note:* For genomic DNA, include tubes 1-4. For other types of DNA, include tubes 1-6. The initial DNA concentration is 100 ng/µl, with final concentrations noted in the boxes above each tube. --- ### 2. Prepare the Reaction Mix Mix the components as per the table below. After combining all elements, briefly vortex the mixture and spin it down.  For Probe-Based Assays:  --- ### 3. Distribute the Reaction Mix Add 20 µl of the prepared reaction mix into the bottom of the blue wells, following the layout in the image below.  --- ### 4. Add the DNA Samples Transfer 5 µl of DNA from the dilution series into the respective wells. Ensure the pipette tip remains submerged as little as possible to avoid contaminating the sides of the wells. The DNA should enter the liquid directly.  *Tip:* Avoid repeatedly pipetting into the wells—mixing occurs naturally during the initial heating phase of the PCR run. Add PCR-grade water to the NTC (non-template control) wells instead of DNA. --- ### 5. Run the Plate Use the PCR cycling protocol outlined below when running the plate on your real-time PCR instrument. Conduct the melt curve analysis according to the instrument's default settings.  For Probe-Based Assays: No melt curve settings are required. --- ### 6. Analyze the Results To approve the master mix, ensure the results align with the criteria listed below. Use the table provided to assess the master mix.  #### Standard Curve Analysis When examining the standard curve, focus on three key parameters: PCR efficiency, R² value, and replicate consistency. - **PCR Efficiency:** Indicates how effectively the target is amplified and should fall between 90-110%. This is a critical parameter. - **R² Value:** Measures how closely the data fits the regression line, ideally ≥ 0.98. - **Replicate Consistency:** Reflects pipetting accuracy; the standard deviation should be ≤ 0.2. #### Melt Curve Assessment In the melt curve, expect a single distinct peak at the intended melt temperature of the product. Note that the melt temperature depends on pH and buffer conditions, so slight variations between mixes are normal. - Ensure no peaks coincide with the NTCs, as this could indicate contamination. - Small secondary peaks are acceptable unless they interfere with the primary product signal. #### Cq Values Cq values should be evaluated alongside PCR efficiency. While higher Cq values may result from low efficiency, they shouldn’t be the sole focus of analysis. Factors like SYBR level, ROX level, and target length can also influence Cq values. #### SYBR Level SYBR levels are less relevant for assessing performance. Adjustments can easily be made by modifying the buffer composition without impacting overall quality. #### Comparing Two Mixes When comparing two mixes, use identical primers and concentrations while preparing separate reaction mixes for each. Distribute samples accordingly and analyze melt curves separately. --- ## Additional Considerations This guide emphasizes simplicity and efficiency. For a comprehensive evaluation, consider other vital factors such as primer/probe design, annealing temperature, inhibitor presence, and appropriate controls. --- ## Extract from Ampliqon Items - **RealQ Plus 2x Master Mix Green Low ROX™ (A324499)** - **RealQ Plus 2x Master Mix for Probe Low ROX™ (A314499)** ## Learn More - Flyers: - "Study of Fast Cycling Real-Time PCR Conditions using RealQ Plus Master Mixes" - "REALQ PLUS MASTER MIXES" - Online Search: Explore real-time master mixes. - Contact Us: - Phone: +33 4 70 03 73 06 - Email: [Insert email here] --- While this guide provides a solid foundation, always tailor evaluations based on specific experimental needs and equipment capabilities. Proper assessment ensures reliable and reproducible results across different applications.

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